Bilirubin IX alpha (BR) is capable of sensitizing the photodynamic hemolysis of human erythrocytes (RBC) in vitro. Although the photolesions which predispose the cells to cation leaks, swelling, and hemolysis occur in the cell membrane, little is known about the molecular nature of these lesions. In previous studies, we showed that irradiation of isolated RBC membranes in the presence of BR produces two major types of damage: (1) lipid peroxidation (LP); and (2) cross-linking of major polypeptides (most notably spectrin), as observed on sodium dodecyl sulfate (SDS)-gel electrophoresis. Cross-linking, which is expected to perturb membrane structure, could play major role in the lytic process. The continuing work will concentrate on the following areas: (A) BR-sensitized photohemolysis will be probed comprehensively. The time course of four events: K ion leakage, Hb leakage, protein cross-linking, and LP will be followed, in an attempt to ascertain the relative importance of cross-linking and LP as determinants of lysis. This work will be paralleled by similar studies in a relatively simple model system, i.e., resealed RBC membranes. The possible involvement of singlet oxygen in photolysis will be examined by using agents or conditions which are "diagnostic" for this species. (B) Attempts will be made to identify at least some of the types of amino acid residues involved in cross-linking, and to determine the chemical nature of the bonds. Two approaches will be used: (1) direct amino acid analysis of the photoproducts; and (2) blocking the reaction by chemical modification of specific residues.